The long range goal of this project is to elucidate fundamental mechanisms of retinal angiogenesis. The overall hypothesis is that retinal angiogenesis depends on molecular interactions between the endothelial cell (EC) derived relaxing factor, nitric oxide (NO), its Ca2+-dependent synthetic enzyme, eNOS and the EC-specific angiogenic factor vascular endothelial growth factor (VEGF). A working model is proposed in which VEGF stimulation of its receptors induces NOS activation and production of NO, which influences EC growth directly by inducing cell proliferation and indirectly by inhibiting VEGF protein expression. This model will be developed and refined by experiments in specific aims designed to test the following specific hypotheses using a combination of cellular and molecular analytical approaches. 1) VEGF- induced growth of retinal ECs is mediated by eNOS production of NO. Experiments using cultured retinal Ecs will determine VEGF effects on NOS activity, NO production and cell growth in the presence and absence of NOS inhibitors and of NO onEC growth. 2) Expression of eNOS and VEGF is regulated by a feed-forward/feed-back relationship in which VEGF promotes increased eNOS expression and NO production which in turn inhibits VEGF expression. These experiments will test NO donor effects on VEGF expression in retinal Ecs and glial cells and VEGF effects on eNOS expression in retinal ECs. 3) eNOS is directly involved in VEGF signal transduction to the nucleus. These experiments will determine the effects of VEGF-stimulation on the subcellular distribution of eNOS, VEGF and its receptors and test the role of NO in VEGF-induction of gene transcription in retinal Ecs. 4) eNOS activity has a critical role in regulating retinal vascular growth and development in vivo. Experiments in eNOS deficient mice will characterize retinal vascular growth and development in relation to expression of inducible NOS (iNOS), constitutive NOS (nNOS) and VEGF. Information gained from these investigations will aid the development of therapies to prevent and control pathological angiogenic processes.